Hi Laura,

how exactly do your stacks look like? I assume they have a black background and on the black background you have dots of two different colors showing the gH2AX and PAX8 foci? Are both types of foci on different channels or do you have both of them in one channel?

For simple quantification of contrasty image stacks that show nicely separated structures you could use the Fiji plug-in "3D Objects Counter". In the "3D Objects Counter Options" you can determine what kind of parameters you want to quantify (e.g. nr. of objects, nr. of voxels per object, volume, surface...).

In case you have the two different types of foci on different channels you could quantify both channels individually to get a number of foci for your gH2AX and a number for your PAX8. Then you merge both channels into one and quantify the number of foci in the merged stack. Then you would only need to calculate the following to get the number of foci that colocalize (if that is the parameter that you want to quantify):

Nr. of colocalizing foci = nr. of gH2AX foci + nr. of PAX8 foci - nr. of foci in the merged stack

In case some of your foci are very close to each other, overlap or are clustered in the stack you could watershed the stack first. This process separates structures which are very close to each other and could be counted as one object by the 3D Objects Counter. Here is a link to a very nice interactive watershed plug-in https://imagej.net/Interactive_Watershed

I just saw that there also is a Fiji plug-in which is called "Colocalization". I never tried it and don't know what it does but maybe it simplifies the workflow that I described above.

Best,

Benjamin

Hi Benjamin,

Thank you so much for your input. Indeed, my stacks have a black background with white objects in the foreground.

I have DAPI, PAX8, and gH2AX foci in different channels.

Regarding the "3D object counter" plugin: do I need to identify the nuclei first, or does the plugin do it automatically?

Best regards,

Laura.

Hi Laura,

the 3D object counter does not automatically identify anything.

The plug-in asks you first to select a threshold to tell the plug-in which pixels/voxels you want to include. The threshold is just a scale showing all the grayscale values of your images. For an 8bit image it ranges from 0-255. A threshold of 70 would then only include pixels that are 70 or brighter. Everything below 70 would be considered as the background. The plug-in then counts all areas in your stack that are selected based on the threshold and gives you some parameters for these. You can also select a minimum size of the objects that you want in your selection. Everything below that size will not be counted. Because the plug-in works with a threshold selection it works best on stacks that have a strong contrast between structures and background.

Regarding your question I assume that you have several cells in your stack and you want to quantify the foci for each individual cell separately? This might make everything a little more complicated. A work-around for this could be that you create subsets of your stack where each subset only shows one cell of your original stack.

Best,

Benjamin